Stereoselective synthesis of the two major metabolites of spironolactone, 3α- and 3β-hydroxy-7α-methylthio-17α-pregn-4-ene-21,17-carbolactone

Article abstract of DOI:10.1016/j.steroids.2007.03.011

7α-Methylthio-3β-hydroxy-17α-pregn-4-ene-21,17-carbolactone and 7α-methylthio-3α-hydroxy-17α-pregn-4-ene-21,17-carbolactone, the two major metabolites of spironolactone, were prepared stereoselectively by exploring different types of reduction reagents. For the 3β-hydroxyl isomer, the application of NaBH₄/CeCl₃·7H₂O gave the best result while for the 3α-hydroxyl isomer, K-selectride seemed to provide the highest stereoselectivity.

Full text of DOI:10.1016/j.steroids.2007.03.011

steroids 7 2 ( 2 0 0 7 ) 569–572
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Stereoselective synthesis of the two major metabolites
of spironolactone, 3␣- and 3␤-hydroxy-7␣-methylthio-17␣-
pregn-4-ene-21,17-carbolactone
Guo Li, Yan Zhang∗
Department of Medicinal Chemistry, Virginia Commonwealth University, 410 North 12th Street,
P.O. Box 980540, Richmond, VA 23298-0540, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
7␣-Methylthio-3␤-hydroxy-17␣-pregn-4-ene-21,17-carbolactone and 7␣-methylthio-3␣-
hydroxy-17␣-pregn-4-ene-21,17-carbolactone, the two major metabolites of spironolactone,
were prepared stereoselectively by exploring different types of reduction reagents. For the
3␤-hydroxyl isomer, the application of NaBH4/CeCl3·7H2O gave the best result while for the
3␣-hydroxyl isomer, K-selectride seemed to provide the highest stereoselectivity.
© 2007 Elsevier Inc. All rights reserved.
Received 10 October 2006
Received in revised form
12 March 2007
Accepted 16 March 2007
Published on line 30 March 2007
Keywords:
Spironolactone
Metabolites
Stereoselective synthesis
1.
Introduction
spironolactone [2]. It is not clear whether they have antimin-
eralocorticoid or antiandrogenic activity, or even induce
Spironolactone (SP) is an aldosterone antagonist, and has
been used for more than thirty years as one of the pre-
ferred diuretics in the treatment of hypertension without
syndrome. As an antiandrogenic agent, spironolactone is
also used to treat hirsutism, acne, and seborrhea [1]. It
has been reported that the major hepatic metabolites of
spironolactone in vivo are canrenone(CAN, 3), 7␣-methylthio-
3-oxo-17␣-pregn-4-ene-21,17-carbolactone (TM, 4), and 7␣-
methylthio-3␣-hydroxy-17␣-pregn-4-ene-21,17-carbolactone
(5). In addition, 7␣-methylthio-3␤-hydroxy-17␣-pregn-4-ene-
21,17-carbolactone (6) has also been identified as one of the
hepatic metabolites of spironolactone in vitro (Scheme 1) [2].
It has been speculated that the isomeric allylic alcohols
5 and 6 may contribute to the pharmacological activities of
mutagenic toxicity. Thus far there is no report on the chemi-
cal synthesis of these two isomers. We hereby report the first
stereoselective synthesis of these two important metabolites
(Scheme 2) for the purpose of further metabolic and pharma-
cological investigation.
2.
Experimental
Melting points were obtained with a Fisher scientific micro
melting point apparatus and were uncorrected. Proton
(300 MHz) and carbon-13 (75 MHz) nuclear magnetic resonance
(NMR) spectra were recorded on a Varian Gemini-300 “Tesla”
spectrometer (300 MHz), with tetramethylsilane (TMS) as the
∗
Corresponding author. Tel.: +1 8048280021; fax: +1 8048287625.
E-mail address: yzhang2@vcu.edu (Y. Zhang).
0039-128X/$ – see front matter © 2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2007.03.011

570
steroids 7 2 ( 2 0 0 7 ) 569–572
Scheme 1 – Proposed metabolism pathway of spironolactone.
internal standard. Infrared spectra (wave numbers in cm−1
)
Found, 411.1950. Anal. Calcd. for: C23H32O3S: C, 71.09; H, 8.30.
Found: C, 70.82; H, 8.51.
were recorded on a Nicolet Avatar 360 FT-IR spectrometer
using KBr discs. TLC analyses were carried out on the Anal-
tech Uniplate F254 plates. GC/MS analysis was performed on
a Hewlett Packard 6890 (Palo Alto, CA). HRMS was determined
by using a Waters Micromass QTof-2(ESI) in positive mode.
Elemental analysis was conducted by the Atlantic Microlab,
Inc. The sample of canrenone was purchased from Atlantic
SciTech Group, Inc. USA.
7␣-Methylthio-3-oxo-17␣-pregn-4-ene-21,17-carbolactone
(4) The compound was prepared according to the reported
procedure from commercial available canrenone [3]. The
product was purified by recrystallization from methanol
yielding 0.80 g (69%) as white powder, m.p. 213–214 ◦C (m.p.
212–213 ◦C [3]). The IR, 1H NMR data and specific rotation data
agreed with the literature values [3]. 13C NMR (75 MHz, CDCl3),
ı: 198.2 (C-3), 176.3 (C-22), 166.5 (C-5), 126.4 (C-4), 95.3 (C-17),
53.1, 47.1, 47.1,46.5, 44.9, 39.5, 38.0, 37.1, 35.1, 34.9, 33.5, 30.8,
30.8, 28,9 21.9, 20.1, 17.8, 13.6. MS (EI): 388, 340, 267, 107, 91.
HRMS (ESI) m/z: calcd for C23H32O3SNa ([M + Na]+), 411.1970;
7␣-Methylthio-3␣-hydroxy-17␣-pregn-4-ene-21,17-
carbolactone (5) [11–13] Compound 4 (300 mg, 0.77 mmol)
in tetrahydrofuran (30 mL) was stirred at −60 ◦C under nitro-
gen protection. Then 1.16 mL 1 M K-Selectride (1.16 mmol) was
added into the solution. After 7 h of stirring at −60 ◦C, 20 ml
water was added into the mixture. The resulting mixture was
concentrated to dryness to remove THF. Then the mixture
was partitioned between dichloromethane (50 mL) and water
(30 mL). The CH2Cl2 layer was dried over anhydrous Na2SO4
and concentrated. The residue was purified by chromatog-
raphy over silica gel (petroleum ether/ethyl acetate = 2/1) to
give 200 mg (68%) 3␣-hydroxyl isomer as colorless amorphous
solid. [˛]2D0 + 13.4 (c 0.02, CHCl3); IR (KBr) ꢀ: 3420 (br, O–H), 1769
(s, C O) cm−1. The proton NMR spectral data agreed with the
reported values [2a]. 13C NMR (75 MHz, CDCl3): ı 176.4 (C-22),
144.3 (C-5), 124.3 (C-4), 95.6 (C-17), 67.21, 48.3, 46.7, 46.4, 45.1,
45.1, 39.6, 39.5, 37.0, 36.3, 35.0, 30.9, 30.9, 28.9, 27.2, 22.1, 20.0,
18.3, 14.0. MS (EI): 390, 372, 342, 325, 105, 91. HRMS (ESI) calcd
for C23H34O3SNa ([M + Na]+), 413.2126; Found, 413.2110. Anal.
Calcd. for: C23H34O3S: C, 70.73; H, 8.77. Found: C, 71.11; H, 9.06.
7␣-Methylthio-3␤-hydroxy-17␣-pregn-4-ene-21,17-
carbolactone (6) [4,5] To the CH3OH (4 mL) solution of
compound
4
(98 mg, 0.25 mmol) and CeCl3·7H2O (93 mg,
0.25 mmol), NaBH4 (20 mg, 0.53 mmol) was added over 5 min
at room temperature. After the mixture was stirred for 20 min,
the reaction was quenched with 1 M HCl. After evaporation of
the majority of CH3OH under reduced pressure, H2O (5 mL)
was added into the residue. The resulting white precipitate
was collected and washed with water. Recrystallization from
methanol yielded 90 mg (92%) 3␤-hydroxyl isomer as white
powder, mp 168–169 ◦C; [˛]D20 + 3.25 (c 0.01 CHCl3); IR (KBr) ꢀ:
3471 (br, O–H), 1761 (s, C O) cm−1. The proton NMR spectral
data agreed with the reported values [2a]. 13C NMR (75 MHz,
CDCl3): ı 176.5 (C-22), 141.8 (C-5), 126.7 (C-4), 95.6 (C-17), 63.51,
53.1, 48.6, 48.6, 47.1, 45.1, 39.7, 36.9, 36.3, 36.3, 35.0, 30.9, 30.9,
28.9, 23.9, 22.1, 20.0, 17.4, 14.5. MS (EI): 390, 372, 342, 325,
267, 105, 91. HRMS (ESI): calcd for C23H34O3SNa ([M + Na]+),
Scheme 2 – The stereo-selective synthesis of the two major
metabolites of spironolactone.

steroids 7 2 ( 2 0 0 7 ) 569–572
571
Table 1 – The reduction of 3-carbonyl group of compound 4
Reagent
Reaction time (h)
% 3␤ isomera
% 3␣ isomera
Conversion (%)b
NaBH4
4
0.5
7
7
24
24
81
92
21
23
40
40
19
7
79
77
60
60
95
99
86
78
66
63
NaBH4/CeCl3·7H2O
K-Selectride
KS-Selectridec
Dihydrosilane-rhodium-(+)diopd
Dihydrosilane-rhodium-(−)diopd
a
The percentages of 3␣ and 3␤ isomer were estimated based on the integrations of the proton at C(4) in the 1H NMR spectra of the crude
reaction mixtures. The integration of the peak with the chemical shift at ı 5.36 was used to calculate the percentage of the 3␤-hydroxyl isomer
in the mixture, whereas that of the peak with the chemical shift at ı 5.55 was used to calculate the percentage of the 3␣-hydroxyl isomer.
Conversion (%) rate was based on the combined yields of the two isomers after separation.
The reaction procedure of KS-Selectride was as same as that of the K-Selectride.
The reaction procedure of dihydrodiphenylsilane-rhodium ( )-diop was adopted from literature [14].
b
c
d
413.2126; Found, 413.2108. Anal. Calcd. for: C23H34O3S: C,
70.73; H, 8.77. Found: C, 70.44; H, 8.71.
The results were summarized in Table 1. By adopting K-
Selectride as the reducing agent, the 3␣-hydroxyl isomer was
the major product. When KS-Selectride was applied, a simi-
lar stereoselectivity was achieved. The chiral reducing agents,
dihydrodiphenylsilane-rhodium( )-diop, also converted com-
pound 4 mainly to the 3␣-hydroxyl isomer, though the
stereoselectivity was not as high as those of the selec-
trides. According to the previous reports, the more hindered
reducing reagents, e.g. the selectride ones, may benefit the
preparation of ␣-isomer over ␤-isomer due to the possi-
bility that the reagent may approach predominantly from
the equatorial side of steroid molecule to afford the axial
alcohol as the main product [18,19]. Our observation in
the preparation of the ␣-isomer with the application of K-
Selectride and KS-Selectride was in agreement with this
speculation. On the other hand, the existence of 7-methylthio
group in the vicinity of the 3-carbonyl group might dimin-
ish the steric hindrance difference between the equatorial
and axial side of the molecule in some degree. Therefore, the
lower stereoselectivity in the preparation of 3␣-isomer was
understandable.
3.
Results and discussion
As the starting material to prepare 3␣- and 3␤-hydroxy-
7␣-methylthio-17␣-pregn-4-ene-21,17-carbolactone (5 and 6),
7␣-methylthio-3-oxo-17␣-pregn-4-ene-21,17-carbolactone (4)
was prepared from the commercial available canrenone (3)
with methylmercaptan in piperidine and methanol around
10 ◦C with a reasonable yield [3].
The well-known procedures to stereoselectively reduce
the 3-carbonyl group in 3-one-4-en steroids to give the 3␤-
hydroxyl isomer include NaBH4/CeCl3·7H2O [4,5], NaBH4 [6],
and NaBH4/Pyridine/Ac2O [7]. LiAlH4 activated template poly-
mers have also been adopted in the literature [8]. Recently a
catalytic hydrogenation method by using Ir-metal particles on
␤ zeolites has been approved to be useful in the preparation of
3␤-hydroxyl isomer too [9]. Some bulky reducing reagents, like
Li(t-BuO)3AlH, may also be applied to prepare the 3␤-hydroxyl
isomer from certain 3-one-4-en steroids [10].
In our case, both NaBH4 and NaBH4/CeCl3·7H2O sys-
tems were tried to prepare 3␤-hydroxyl isomer (6). The
NaBH4/CeCl3·7H2O system reduced the 3-carbonyl group with
very high stereoselectivity and the ratio of 3ˇ/3˛ isomer
was about 13:1. Using NaBH4 as the reduction reagent, the
ratio of 3ˇ/3˛ isomer was about 4:1 (Table 1). Apparently,
the NaBH4/CeCl3•7H2O system was more promising. Another
advantage of the NaBH4/CeCl3·7H2O system was the shorter
reaction time, which was believed to be beneficial to the stere-
oselectivity.
In summary, for the synthesis of 3␤-hydroxyl isomer (6),
NaBH4/CeCl3·7H2O method gave higher stereoselectivity, and
for the preparation of 3␣-hydroxyl isomer (5), K-selectride
seemed to be the best choice.
Acknowledgement
The research conducted was partially supported by A.D.
Williams Foundation Research Fund (6-46920).
r e f e r e n c e s
The reduction reagent to prepare the 3␣-hydroxyl
isomer from 3-one-4-en steroids include K-Selectride
[11–13], KS-Selectride [12], dihydrosilane-rhodium-(+)diop
and dihydrosilane-rhodium-(−)diop [14], and aluminum
isopropoxide-isopropyl alcohol [15]. Controversially, Li(t-
BuO)3AlH was also applied to achieve the 3␣-isomer for some
substrates [16,17].
In order to stereoselectively prepare the 3␣-hydroxyl
isomer (5), several reduction reagents, e.g. K-Selectride,
KS-Selectride, dihydrosilane-rhodium-(+)-diop, and dihydro-
silane-rhodium- (−)-diop, have been applied on our substrate.
[1] (a) Wolf RL, Mendlowitz M, Roboz J. Treatment of
hypertension with spironolactone: double-blind study. JAMA
1966;198:1143–9;
(b) Jeunemaitre X, Chatellier G, Kreft-Jais C. Efficacy and
tolerance of spironolactone in essential hypertension. Am J
Cardiol 1987;60:820–5;
(c) Smith AGE. Spironolactone in the long-term
management of patients with congestive heart failure. Curr
Med Res Opin 1980;7:131–6;

572
steroids 7 2 ( 2 0 0 7 ) 569–572
(d) Ikram H, Webster MWI, Nicholls MG, Lewis GRJ, Richards
[9] De Bruyn M, Coman S, Bota R, Parvulescu VI, De Vos DE,
Jacobs PA. Chemoselective reduction of complex
␣,␤-unsaturated ketones to allylic alcohols over Ir-metal
particles on ␤ zeolites. Angew Chem 2003;42:5333–6.
[10] Loughhead DG. Preparation and acid-catalyzed
rearrangement of a C-17 isopropylidene and a C-17
isopropenyl sterol. J Org Chem 1985;50:3931–4.
[11] Gondos G, Orr JC. Stereoselective and regioselective
reduction of steroid ketones by potassium
tri(R,S-s-butyl)borohydride. J Chem Soc Chem Commun
1982:1239–40.
[12] Wiebe JP, Deline C, Buckingham KD. Synthesis of the allylic
gonadal steroids, 3␣-hydroxy-4-pregnen-20-one and
3␣-hydroxy-4-androsten-17-one, and of
3␣-hydroxy-5␣-pregnan-20-one. Steroids 1985;45:39–51.
[13] Gondos G, McGirr LG, Jablonski CR, Snedden W, Orr JC. The
reduction of steroid 2␣-fluoro-4-en-3-ones. J Org Chem
1988;53:3057–9.
[14] Gondos G, Orr JC. Reduction of steroid 17-ketones by
enantiomeric chiral reducing agents. J Chem Soc Chem
Commun 1982:1238–9.
[15] Wiebe JP, Dave V, Stothers JB. Synthesis and characteristics
of allylic 4-pregnene-3,20-diols ound in gonadal and breast
tissues. Steroids 1986;47:249–59.
[16] Nicoletti D, Ghini AA, Furtmueller R, Sieghart W, Dodd RH,
Burton G. Synthesis and GABAA receptor activity of
6-oxa-analogs of neurosteroids. Steroids 2000;65:349–56.
[17] Veleiro AS, Rosenstein RE, Jaliffa CO, Grilli ML, Speroni F,
Burton G. Synthesis and GABAA receptor activity of a
6,19-oxido analogue of pregnanolone. Bioorg Med Chem Lett
2003;13:343–6.
AM, Croziec IG. Combined spironolactone and
converting-enzyme inhibitor therapy for refractory heart
failure. Aust N Z Med 1986;16:61–3;
(e) Huffman DH, Kampmann JP, Hignite CE, Azarnoff DL.
Gynecomastia induced in normal males by spironolactone.
Clin Pharmacol Ther 1978;24:465–73;
(f) Shapiro G, Evron S. A novel use of spironolactone:
treatment of hirsutism. J Clin Endocrinol Metab
1980;51:429–32;
(g) Cumming DC, Yang JC, Rebar RW, Yen SCC. Treatment of
hirsutism with spironolactone. JAMA 1982;247:1295–8.
[2] (a) Los LE, Pitzenberger SM, Ramjit HG, Cod Dington AB,
Colby HD. Hepatic metabolism of spironolactone: production
of 3-hydroxy-methylthio metabolites. Drug Metab Dispos
1994;22:903–8;
(b) Ricardo G, Lucia N, Ricardo C, Miguel V, Juan T, Eva D.
Spironolactone and its main metabolite canrenoic acid block
hKv1.5, Kv4. 3 and Kv7. 1 + minK channels. Br J Pharmacol
2005;146:146–61.
[3] Karim A, Brown EA. Isolation and identification of novel
sulfur-containing metabolites of spironolactone. Steroids
1972;20:41–62.
[4] Luche J-L, Rodrigues-Hahn L, Crabbe P. Reduction of natural
enones in the presence of cerium trichloride. JCS Chem
Comm 1978:601–2.
[5] Cui JG, Lin CW, Zeng LM, Su JY. Synthesis of
polyhydroysterols (III): synthesis and structural elucidation
of 24-methylenecholest-4-en-3␤,6␣-diol. Steroids
2002;67:1015–9.
[6] Freixa Z, Pereira MM, Bayon C, Silva AMS, Salvador JAR, Beja
AM, Paixao JA, Ramos M. Diastereoselective
hydroformylation of ꢁ4-steroids with rhodium-phosphite
catalysts. Tetrahedron: Asymmetry 2001;12:1083–7.
[7] Nahar L, Turner AB. Synthesis of 3␤,
[18] Brown HC, Krishnamurthy S. Lithium
tri-sec-butylborohydride. A new reagent for the reduction of
cyclic and bicyclic ketones with super stereoselectivity. A
remarkably simple and practical procedure for the
conversion of ketones to alcohols in exceptionally high
stereochemical purity. J Am Chem Soc 1972;94:7159–61.
[19] Hutchins RO. Conformational control. An important factor
in the stereoselective reduction of ketones by bulky hydride
reagents. J Org Chem 1977;42:920–2.
6␣-dihydroxy-5␣-cholan-23-one. Tetrahedron
2003;59:8623–8.
[8] Bystroem SE, Boerje A, Akermark B. Selective reduction of
steroid 3- and 17-ketones using LiAlH4 activated template
polymers. J Am Chem Soc 1993;115:2081–3.